JP2630770B2 - Modification of phospholipids - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method of transesterifying phospholipids by allowing a phospholipid and a fatty acid or a lower alcohol ester of a fatty acid to act on an enzyme having a decomposing action of toglyceride. The present invention relates to a method for modifying lipid.

(Prior Art) Phospholipids are formed by bonding a hydrophilic phosphorus-containing compound such as phosphorylcholine, phosphorylethanolamine, or phosphorylinositol with glycerol and a fatty acid. Functional properties such as activity are largely affected by the type of bound fatty acid.

As one of means for adjusting the physical properties and the like by changing the type of fatty acid bound to the glycerol residue, there is a method of transesterifying a fatty acid by utilizing the action of an enzyme having transesterification ability. Many studies have been made on the modification of fats and oils (triglycerides) by this method, and there are many patent applications relating to this (for example, Japanese Patent Application Laid-Open No. 61-149097, and The main patent applications and their evaluations are described).

On the other hand, with respect to phospholipids, enzymatic modification causes an enzyme such as snake venom folipholipase or pancreatin, which has the ability to break the acyl bond of phospholipids, and decomposes a part of the phospholipids to form lysophospholipids that are hydrophilic or hydrophilic. Methods for improving the emulsifying properties are known (for example, Japanese Patent Application No. 53-7151).
7).

However, regarding the reaction of fatty acids of phospholipids by enzymes, Z. Naturforsch
Vol. 25b, pp. 581-586 (1970) only reports that HPFranck et al. Have found that folipholipase A has the ability to bind fatty acids to lyso-type phospholipids. is there. However, later reports revealed that this was also a false conclusion due to experimental deficiencies [J. Goerke et al .: Biochimica et Biophysica Acta.
ca et Biophysica Acta) No. 248, pp. 245-253 (197
1)].

As described above, an enzyme having a transesterification effect that changes the type and position of a fatty acid bound to a phospholipid has not been known at all.

(Problems to be Solved by the Invention) In the present invention, in order to investigate the reactivity of an enzyme with phospholipids, the enzyme activation treatment and the reaction conditions were examined in detail, and as a result, appropriate reaction conditions were set. Thus, the present inventors have found that a certain enzyme exerts a transesterification effect on phospholipids, and based on this finding, seek to pave the way for the practical application of phospholipid modification by transesterification.

(Constitution of the Invention) The present invention provides a method for modifying a phospholipid, which comprises subjecting a system in which a phospholipid and a fatty acid or a fatty acid lower alcohol ester are present to an enzyme having a triglyceride-decomposing action to effect transesterification of the phospholipid. Is the law.

Phospholipids to be subjected to transesterification by the enzyme of the present invention include phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and other simple phospholipids, for example, soybean lecithin, yolk lecithin and other commercial lecithins of animal and plant origin, and these phospholipids Is a so-called lyso-type phospholipid in which a part of the bound fatty acid has been removed from the oil, and is obtained in a degumming step which is one of the purification steps of vegetable oil. There is a so-called extracted soap scum containing a phospholipid component.

As the fatty acid, a linear saturated fatty acid, unsaturated fatty acid, or highly unsaturated fatty acid having about 8 to 24 carbon atoms can be used, and examples thereof include palmitic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid. The lower alcohol ester of a fatty acid refers to an ester compound of the above fatty acid and a linear saturated monohydric alcohol having 1 to 6 carbon atoms. Examples thereof include methyl palmitate, methyl stearate, methyl linoleate, ethyl palmitate, and stearic acid. ethyl,
Ethyl linoleate and the like.

Enzymes that act on these phospholipids and fatty acids or fatty acid lower alcohol esters to change the type and position of the fatty acids bound to the phospholipids, that is, enzymes used in the present invention as enzymes having a transesterification effect include, for example, Alpergillus spp. Microorganisms belonging to the genera Rhizopus, Mucor, Pseudomonas, Arisrobacter, Candida, Chromobacterium, Penicillium are produced, or there are enzymes having a triglyceride-degrading action derived from the pancreas. Commercially available as lipase.

Examples of microorganisms belonging to the above genera include Aspergillus niger in Aspergillus.
r) or Aspergillus wenchi
nti) In the genus Rhizopus, Rhizopus delemar
elemar), Rhizopus javani
cus), Rhizopus niveus, Mucor mihei for the genus Mucor, Mucor javanicus for the genus Mucor, and Pseudomonas fluorescens for the genus Pseudomonas
domonus fluorescence), Pseudomonus fragi (Pseudomonus fragi), and in the genus Aliceobacter, Ahrismobacter urea faciliens (Arthrobaoter ur)
eafaciens), Candida cylindracea for Candida, and Chromobacterium biscosum for Chromobacterium
ctrium viscosum) and Penicillium cyclopium in the genus Penicillium. Enzymes produced by these are known to have lipase activity, and many of them are already commercially available. The present invention has newly found that these known enzymes have a phospholipid transesterification effect, and found that they can be suitably used in the present invention, thereby completing the present invention.

These enzymes were known to be used for transesterification of triglycerides. However, phospholipids differ greatly in polarity from triglycerides and alcohol esters, and it has heretofore been considered that it is difficult to perform transesterification using an enzyme.

The reaction is conducted by adding a phospholipid and a fatty acid or a fatty acid lower alcohol ester to a nonpolar solvent such as hexane in an amount of 1 to 50% by weight.
Water containing an amount of enzyme equivalent to 10 to 50,000 units of lipase activity measured by the method of Yamada et al. {Journal of the Japanese Society of Agricultural Chemistry 36 , 860 (1962)} per gram of phospholipid. Alternatively, a buffer solution is added, and the reaction solution is shaken at 20 to 26 ° C, more preferably at 25 to 40 ° C for 5 to 72 ° C.
Let react for hours. Water or buffer containing the enzyme is added in an amount exceeding the saturated water content of the organic solvent as shown in the examples. The ratio of the phospholipid to the fatty acid or fatty acid lower alcohol ester in the reaction system is desirably in the range of 1: 0.2 to 1: 5.

The reaction in the present invention is performed as a two-phase reaction between an organic solvent phase containing raw materials and products and an aqueous phase containing enzymes, since an amount of water or buffer exceeding the saturated water content of the organic solvent is added. Done.

As a means for increasing the activity of the enzyme, a celite-based support and an activating agent such as ethanol and propanol can be appropriately added to the reaction system. In addition, bioreactors that have been variously devised for the enzymatic reaction can also be used, and in order to suppress hydrolysis, a device that removes water from the reaction system as much as possible within a range of a two-phase reaction is as follows. It is beneficial to improve the yield.

The enzymes used in the present invention do not require chemical modification.

After a predetermined time, the reaction solution is inactivated by heating or the like, if necessary, to deactivate the lipase, and concentrated by a suitable concentrator as needed, and further subjected to appropriate drying means such as spray drying and freeze drying, and concentrated. Or use a dry product. Further, the reaction product is extracted and separated from the reaction system by using an appropriate solvent such as benzene or chloroform at any of the above-mentioned stages, and after performing solvent separation, purification such as column treatment as necessary, the solvent is distilled off. If this is the case, a higher-purity modified phospholipid can be obtained.

(Examples) Hereinafter, examples of the present invention will be described. In the present invention, an enzyme commercially available as a lipase can be used.
Those skilled in the art can easily understand that other enzymes described in the specification can be similarly used in the present invention based on the examples shown in the following examples.

Example 1 An enzyme derived from Rhizopus delemar (manufactured by Tanabe Seiyaku Co., Ltd.
"Talipase", 50 mg dispersed in 0.1 ml of TES buffer (PH6.5), n-hexane solution containing 1.6% (w / v) dipalmitoyl phosphatidylcholine and 1.6% (w / v) oleic acid 0.9 ml was added, and the reaction mixture was sealed and reacted at 35 ° C. for 48 hours in a reciprocating shaker.

After drying the reaction mixture, the oil content is extracted with chloroform, the remaining amount of phosphatidylcholine is determined by Iatroscan, and the phosphatidylcholine fraction is separated by silica gel thin layer chromatography (developing solvent: chloroform: methanol: water 60: 30: 3) The fatty acid composition was examined by gas chromatography.

As a result of analysis, the residual rate of phosphatidylcholine was 62%,
Before the reaction, the fatty acid composition was 99.7% of palmitic acid and 0.1% of oleic acid, whereas after the reaction, it was 80.6% of palmitic acid and 19.4% of oleic acid, and a transesterification reaction of phospholipid was clearly observed. .

Example 2 2 g of an enzyme derived from Rhizopus delemar (“Taripase”, Tanabe Seiyaku Co., Ltd.) was dispersed in 2 ml of TES buffer (PH6.5), and soybean phospholipid (Sternbar P manufactured by Star Chemie, Germany) was dispersed.
M) 2 g, oleic acid 2 g and n-hexane 18 ml were added and reacted at 35 ° C. for 24 hours in a rotary shaker.

The phosphatidylcholine (PC) and phosphatidylethanolamine (PE) fractions were collected from the phospholipid component in the reaction solution by thin-layer chromatography according to Example 1,
Each fatty acid composition was analyzed by gas chromatography.

The results were as shown in Table 1, phosphatidylcholine,
Both phosphatidylethanolamines were transesterified, and the content of oleic acid increased by about 13%, and the content of saturated fatty acids such as palmitic acid and stearic acid decreased significantly.

Example 3 Phospholipids before and after the reaction were performed in the same manner as in Example 2 except that an enzyme derived from the genus Pseudomonas ("Lipase P" manufactured by Amano Pharmaceutical Co., Ltd.) was used and the fatty acid was changed to methyl oleate. Was examined for fatty acids.

The results are as shown in Table 2, where the oleic acid content increased in both the phosphatidylcholine and the phosphatidylethanolamine fraction. In this example, unlike Example 2, a phenomenon in which only specific fatty acids were significantly reduced was not observed.

Example 4 The fatty acid composition of phosphatidylcholine after the reaction was examined in the same manner as in Example 1, except that 1.3 mg of pancreatic lipase [from Boehringer Mannheim, derived from pig pancreas] was used as the enzyme.

The fatty acid composition of the phosphatidylcholine after the reaction was 89.2% for palmitic acid and 10.3% for oleic acid, and a transesterification reaction was observed.

(Effects of the Invention) The present invention has opened the first way to the modification of phospholipids by transesterification using enzymes.

With the development of phospholipid transesterification technology using enzymes, it has become possible to arbitrarily adjust the physical properties such as the melting point and fluidity of phospholipids, and in the food industry and other industries,
The adjustment of the phospholipid optimal for the use becomes possible. Also,
Phospholipids are attracting attention as carriers for various drugs and enzymes. It is also a material for preparing liposomes. By using a phospholipid whose fatty acid composition has been changed by the method of the present invention, its physiological action can be significantly enhanced.

Since the present invention uses an enzyme, it does not decompose highly unsaturated fatty acids as compared with a chemical synthesis method, and introduces a desired fatty acid into a specific site of a phospholipid utilizing the specificity of the enzyme. Yes, there are advantages.

In the present invention, since there is no need to use a chemically modified enzyme for which safety has not been established, safety does not pose a problem even if the enzyme remains in the product.

In addition, the method of the present invention is carried out as a two-phase reaction between an organic solvent system containing the raw materials and the product and an aqueous phase containing the enzyme, so that the product and the enzyme are easily separated, and thus the product is easily produced. This is also advantageous in terms of product purification and enzyme recycling.

Claims (1)

(57) [Claims] 1. A method for modifying phospholipids, wherein a phospholipid and a fatty acid or a lower alcohol ester of a fatty acid are used as substrates, and an enzyme having an action of decomposing triglyceride is allowed to act on the substrate to carry out fatty acid transesterification of the phospholipid. .